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Gene defect and mutant ALK kinase in human entity tumour

A residue and amino acid technology, applied in the field of diagnosis and treatment, cancer detection, can solve problems such as side effects and troubles

Active Publication Date: 2009-06-24
CELL SIGNALING TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of this drug represents a significant advance over conventional therapies for CML and ALL (chemotherapy and radiation), which are plagued by well-known side effects and often have limited efficacy because they cannot specifically target To the basic cause of cancer (malignant tumor)

Method used

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  • Gene defect and mutant ALK kinase in human entity tumour
  • Gene defect and mutant ALK kinase in human entity tumour
  • Gene defect and mutant ALK kinase in human entity tumour

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0294] Identification of ALK kinase activity in solid tumors by global phosphopeptide profile

[0295] a. Distribution of human NSCLC cell lines

[0296] Kinase activation in 22 human NSCLC cell lines, including H2228, was examined using a recently described powerful technique for the isolation and mass spectrometric characterization of modified peptides from complex mixtures (the "IAP" technique, see Rush et al., supra). global phosphorylation profile. Using phosphotyrosine-specific antibodies ( Beverly, MA, 2003 / 04 Cat. #9411 ) implemented the IAP technique to isolate and subsequently characterize phosphotyrosine-containing peptides from extracts of NSCLC cell lines.

[0297] Specifically, the IAP approach was used to facilitate the identification of tyrosine kinases responsible for protein phosphorylation in each NSCLC cell line. In particular, atypical or abnormal kinase activity is considered.

[0298] cell culture

[0299] All cell culture reagents were purchas...

Embodiment 2

[0322] Isolation and sequencing of three ALK fusion genes

[0323] a. Sequencing in Human NSCLC Cell Lines

[0324] Given the high phosphorylation levels of ALK kinase detected in the NSCLC cell line H2228, rapid amplification of the 5' end of the cDNA at the sequence encoding the kinase domain of ALK was performed in order to determine the presence of chimeric ALK transcripts.

[0325] Rapid amplification of complementary DNA ends

[0326] RNeasy Mini Kit (Qiagen) was used to extract RNA from H2228 cell line. DNA was extracted by using DNeasy Tissue Kit (Qiagen). The cDNA ends were rapidly amplified with the aid of the 5' RACE system (Invitrogen), with primers ALK-GSP1 for cDNA synthesis and ALK-GSP2 and ALK-GSP3 for nested PCR reactions.

[0327] 5'RACE

[0328] Figure 5 (Panel A) shows the detection of the EML4-ALK fusion gene (short variant) by 5' RACE and the detection of the PCR amplification product after 2 rounds. PCR products were purified with a PCR purifi...

Embodiment 3

[0352] Inhibition of Growth of ALK Fusion-Expressing Mammalian Solid Tumors Using siRNA

[0353] To confirm that truncated / fusion forms of ALK are driving cell growth and survival in the NSCLC cell line H2228 as well as in NSCLC tumor samples from patients CS010 / 11, CS045, and CS110, siRNA (relative to ALK) inhibition of these cells can be examined and tumor growth capacity.

[0354] ALK SMARTpool siRNA duplexes (proprietary target sequence - data not shown) can be purchased from, eg, Dharmacon Research, Inc. (Lafayette, CO). Non-specific SMARTpool siRNA was used as a control. Cells were transfected with siRNA by means of electroporation. Briefly, 2 × 10 7 Each cell (H2228) was pulsed once (20ms; 275V, K562 20ms; 285V), incubated at room temperature for 30 minutes, and then transferred to a T150 flask containing 30ml RPMI-1640 / 10% FBS.

[0355] With CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (Promega) to determine the number of viable cells. IC was calc...

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Abstract

In accordance with the invention, novel gene deletions and translocations involving chromosome 2 resulting in fusion proteins combining part of Anaplastic Lymphoma Kinase (ALK) kinase with part of a secondary protein have now been identified in human solid tumors, e.g. non-small cell lung carcinoma (NSCLC). Secondary proteins include Echinoderm Microtubule- Associated Protein-Like 4 (EML-4) and TRK- Fusion Gene (TFG). The EML4-ALK fusion protein, which retains ALK tyrosine kinase activity, was confirmed to drive the proliferation and survival of NSCLC characterized by this mutation. The invention therefore provides, in part, isolated polynucleotides and vectors encoding the disclosed mutant ALK kinase polypeptides, probes for detecting it, isolated mutant polypeptides, recombinant polypeptides, and reagents for detecting the fusion and truncated polypeptides. The disclosed identification of this new fusion protein enables new methods for determining the presence of these mutant ALK kinase polypeptides in a biological sample, methods for screening for compounds that inhibit the proteins, and methods for inhibiting the progression of a cancer characterized by the mutant polynucleotides or polypeptides, which are also provided by the invention.

Description

[0001] related application [0002] This application claims priority to and benefit of currently pending USSN 60 / 792,364, filed April 14, 2006, the entire disclosure of which is hereby incorporated by reference. technical field [0003] The present invention generally relates to proteins and genes associated with cancer, and to the detection, diagnosis and treatment of cancer. Background technique [0004] Many cancers are characterized by disruptions in cellular signaling pathways, which lead to abnormal control of cellular processes, or to uncontrolled growth and proliferation of cells. These disruptions are often caused by changes in the activity of specific signaling proteins such as kinases. These cancers include solid tumors such as non-small cell lung cancer (NSCLC). In the United States, NSCLC is the leading cause of cancer death and accounts for approximately 87% of all lung cancers. There are approximately 151,000 new cases of NSCLC each year in the United State...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07K1/00A61K38/00C07K16/00G01N33/53C12Q1/68
CPCC12Q2600/178G01N33/57423C12Q2600/156C07K14/82C12N9/1205C07K2319/00C12Q1/6886A61K38/00C12Q2600/136A61P35/00C07K14/47C12N9/12C12N15/62C07K19/00
Inventor 克拉里萨·里科瓦赫伯特·哈克劳拉·沙利文顾挺磊安东尼·波塞马托郭爱兰琼·麦克尼尔俞建
Owner CELL SIGNALING TECHNOLOGY
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